1. Field of the Invention
The present invention relates generally to detection of one or more analytes in a sample and/or the magnitude of or changes in physical properties of a sample. More particularly, it concerns pore-subunit polypeptides covalently linked to one or more sensing moieties, and the use of these modified polypeptides to detect and/or measure analytes or certain physical characteristics within a given sample.
2. Description of Related Art
The examination and manipulation of individual molecules is a thriving area of research. Single molecule detection methods, which include electrical recording (Hladky and Haydon, 1970; Sakmann and Neher, 1995), optical spectroscopy (Moerner and Orrit, 1999; Weiss, 1999) and force measurements (Mehta et al., 1999), can provide structural and functional information that is often difficult or impossible to obtain by conventional techniques, which measure the properties of large ensembles of molecules. Recent accomplishments include observations of the movement of individual atoms and small molecules (Gimzewski and Joachim, 1999), the movement of linear and rotary motor proteins (Mehta et al., 1999), the turnover of individual enzymes (Xie and Lu, 1999) and the unfolding and refolding of proteins (Mehta et al., 1999).
In the area of biosensors, progress has been made in developing protein channels and pores as sensor elements (Ziegler and Gopel, 1998; Bayley, 1999). According to this concept, analyte molecules modulate the ionic current passing through the pores under a transmembrane potential. For example, binding sites can be engineered into pores expressly for capturing analyte molecules, which act as partial channel blockers. Stochastic sensing, which uses currents from single pores, is an especially attractive prospect (Braha et al., 1997; Gu et al., 1999). The approach yields both the concentration and identity of an analyte, the latter from its distinctive current signature. Using certain types of stochastic sensing, the inventors have succeeded in detecting divalent metal ions (Braha et al., 1997) and a variety of organic molecules (Gu et al., 1999).
Despite the initial development of stochastic sensing, there remains in the art a need for sensing elements and systems that respond to a wider variety of analytes. The development of stochastic sensing components and systems that permit interactions with sensor elements that would not normally occur with the existing methodology would represent a significant advance in the art.